CN110273023B - 肉桂及其近缘植物的鉴定或鉴别方法,和该方法所用的引物及试剂盒 - Google Patents
肉桂及其近缘植物的鉴定或鉴别方法,和该方法所用的引物及试剂盒 Download PDFInfo
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Abstract
本发明的目的在于提供一种高准确度鉴别肉桂(Cinnamomum cassia)及其近缘植物的方法,并提供了该方法所用的引物及试剂盒。所述近缘植物为锡兰肉桂(Cinnamomum zeylanicum)、香樟(Cinnamomum camphora)和沉水樟(Cinnamomum micranthum)植物。该方法通过检测上述四种植物叶绿体基因组的序列并比对,寻找肉桂等四种植物叶绿体基因组的差异,为科学地鉴别该四种植物提供科学依据。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种肉桂及其近缘植物的鉴定或鉴别方法,和该方法所用的引物及试剂盒。
背景技术
DNA分子鉴定技术是利用分子生物学技术中药材或植物不同物种中特异性的DNA序列进行分析鉴定,从而达到从分子水平上对物种和个体进行鉴定的目的。它作为近代科学技术发展的生要方法已经被广泛应用到各种物种的分类鉴定并不断有新的技术应用于该领域。2010年版《中国药典》收录了川贝母、乌梢蛇、蕲蛇的分子鉴别方法,2015年版《中国药典》建立了《中药材DNA条形码分子鉴定法指导原则》,由此可见,中药的分子鉴定逐渐成为一种先进可行的中药材鉴别方法。在这些技术中PCR技术为基础应用的较多,根据原理的不同,如随机扩增多态性DNA(random amplification polymorphic DNA,RAPD)、简单序列重复区间(inter simple sequence repeat,ISSR)、简单重复序列(simple sequencerepeat,SSR)、扩增片段长度多态性(amplified fragment length polymorphism,AFLP)、单核苷酸多态性(single nucleotide polymorphism,SNP)等方法均广泛用于中药材的鉴别。随着测序技术的发展,目前已由多态性鉴别发展为DNA条形码鉴定。
近年来,中药DNA条形码鉴定,即从基因组标准化区间中得到的可以用于鉴别、发现、区分物种以及给未确定的个体定种的一段基因短片段的方法,该方法在中药材鉴别中得到快速发展。
肉桂为樟科樟属肉桂(Cinnamomum cassia)的干燥树皮,具有广泛的药用价值和食品调料,具有补火助阳,引火归元,散寒止痛,温通经脉的作用,临床主要用于阳痿宫冷,腰膝冷痛,痛经闭经等。市场需求量大,品种较繁杂,其中锡兰肉桂(Cinnamomumzeylanicum)为常见的外来品种,主要产地为斯里兰卡,近几年在我国市场上分布也较广泛,可做肉桂药用。香樟(Cinnamomum camphora)是我国南方和西南地区广泛分布的药用植物,木材及根、枝、叶可提取樟脑和樟油,樟脑和樟油供医药及香料工业用,其皮常和肉桂皮经常混淆。沉水樟(Cinnamomum micranthum)是我国樟科樟属的重要速生经济树种,沉水樟叶所含的樟脑油比重大于水,故而得名,植株可提取芳香油,主含黄樟油素,是工业和医药用的重要原料,树皮也有混肉桂用的。由于上述四种相近的药用植物在外观形状颜色及气味上相近,因而现有技术中常常存在将它们混用的情况,不利于规范用药及保障肉桂类食品的安全。
有鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种高准确度鉴别肉桂和近缘植物的方法,并提供了该方法所用的引物及试剂盒。
检测肉桂叶绿体基因组的序列,通过比对肉桂(Cinnamomum cassia)常见的近缘种植物锡兰肉桂(Cinnamomum zeylanicum)、香樟(Cinnamomum camphora)和沉水樟(Cinnamomum micranthum)植物叶绿体基因序列,寻找肉桂等四种植物叶绿体基因组的差异,为科学地鉴别该四种植物提供科学依据。
具体的,本发明涉及一种引物组合产品,其包括下列引物对:
引物对P1:SEQ ID NO:1-2;
引物对P2:SEQ ID NO:3-4;
引物对P3:SEQ ID NO:5-6;以及
引物对P4:SEQ ID NO:7-8。
上述引物对可用于扩增肉桂、锡兰肉桂、香樟和沉水樟植物的4段特异区段,进而可通过位于这些特异区段上的SNP位点对上述植物进行区分。
本发明还涉及一种试剂盒,其含有如上所述的引物组合产品。
本发明还涉及肉桂及其近缘植物的鉴定或鉴别方法,所述近缘植物为锡兰肉桂、香樟和沉水樟植物,所以方法包括:
a)获取待检测植物的基因组DNA或线粒体DNA;
b)使用如上所述的引物组合产品或如上所述的试剂盒对a)所得DNA进行扩增;
c)将扩增所得片段测序并将测序结果与肉桂特异DNA分子标记矩阵进行比对,若与所述矩阵中某物种总相似性大于95%则判定所述待检测植物为该物种;
所述矩阵为:
表I引物对P1所扩增区域包含的SNP
表II引物对P2所扩增区域包含的SNP
续表II
表III引物对P3所扩增区域包含的SNP
续表III
表IV引物对P4所扩增区域包含的SNP
本发明申请人经过创造性的劳动和大量的试验,得到了能够用于鉴定或鉴别肉桂、锡兰肉桂、香樟和沉水樟的引物和方法。本发明为肉桂和近缘植物的鉴别提供指导,为规范用药及保障肉桂类食品的安全提供强有力的技术支持。
具体实施方式
本发明涉及一种引物组合产品,其包括下列引物对:
引物对P1:SEQ ID NO:1-2;
引物对P2:SEQ ID NO:3-4;
引物对P3:SEQ ID NO:5-6;以及
引物对P4:SEQ ID NO:7-8。
所述引物对可以根据本领域的现有技术合成,也可以委托专业的公司制备。
该组合产品可以将SEQ ID NO:1-8所示的核苷酸分别储存于不同的容器,例如EP管中,也可以每对混合存放,优选按使用时的比例混合。
根据本发明的一方面,本发明还涉及一种试剂盒,其含有如上所述的引物组合产品。
在一些实施方式中,所述试剂盒还含有DNA提取体系、PCR反应缓冲液、水、DNA聚合酶以及分子量marker中的一种或多种。
在一些实施方式中,所述DNA聚合酶选自Taq、Bst、Vent、Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、ES4DNA聚合酶以及Klenow片段中的任一种。
在一些实施方式中,所述水为无核酸酶的水,例如蒸馏水(Distilled Water)、去离子水(Deionized Water)或反渗水(Reverse osmosis Water)。
在一些实施方式中,所述试剂盒还含有测序所需试剂。
根据本发明的一方面,本发明还涉及肉桂及其近缘植物的鉴定或鉴别方法,所述近缘植物为锡兰肉桂、香樟和沉水樟植物,所以方法包括:
a)获取待检测植物的基因组DNA或线粒体DNA;
b)使用如上所述的引物组合产品或如上所述的试剂盒对a)所得DNA进行扩增;
c)将扩增所得片段测序并将测序结果与肉桂特异DNA分子标记矩阵进行比对,若与所述矩阵中某物种总相似性大于95%(例如96%、97%、98%、99%、99.9%)则判定所述待检测植物为该物种;
所述矩阵如如表I~IV所示。
该方法可用于鉴定肉桂、锡兰肉桂、香樟和沉水樟;亦可用于上述四种植物中的任两种或多种的鉴别。
作为待检样品,无特殊限制,只要是有可能包含肉桂和近缘植物的样品即可。例如,肉桂和近缘植物的破碎物、粉碎物、干燥物和提取物,包含这些物质的生药和汉方药,以及包含这些生药和汉方药的医药品、食品、饮料和化妆品等。
在一些实施方式中,在各引物对进行扩增时,退火温度均为49℃~53℃,例如50℃、51℃、52℃。
在一些实施方式中,所述测序选自高通量测序。
在一些实施方式中,所述测序选自Sanger测序。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
本实施例通过以下技术方案予以实现:
S1.收集肉桂叶片;
S2.提取肉桂叶片DNA;
S3.对肉桂叶片DNA进行Illumina DNA文库构建;
S4.Illumina高通量测序;
S5.组装肉桂叶绿体基因组序列;
S6.检索下载肉桂近缘物种序列,如锡兰肉桂,香樟,沉水樟叶绿体基因组序列;
S7.软件对比分析肉桂,锡兰肉桂,香樟,沉水樟叶绿体基因组序列;
S8.设计特异引物;
S9.构建肉桂特异DNA分子标记矩阵和评分标准;
S10.验证:收集近缘种锡兰肉桂、香樟和沉水樟叶片;
S11.提取锡兰肉桂、香樟和沉水樟叶片总DNA;
S12.进行PCR扩增与序列测序,进行物种鉴定对比。
S10-S12为序列验证。
步骤S3所述Illumina DNA文库构建的方法为:依照Illumina文库构建流程,将所述步骤S2中所述总DNA构建为片段大小400~600bp的双末端高通量测序文库;
步骤S7所述对比分析叶绿体序列的方法为:使用序列比对分析软件(如:clustal,muscle,blast等),将步骤S5所述的肉桂叶绿体基因组DNA序列与近缘物种叶绿体基因组序列比对,找出存在差异的区域。以下序列比对结果中,无“*”标记的位置为差异区域。
特异区段1
Cinnamomum_camphora ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomum_micranthum ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomum cassia ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomumzeylanicum ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
************************************************************
Cinnamomum_camphora ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomum_micranthum ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomum cassia ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomumzeylanicum ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
************************************************************
Cinnamomum_camphora GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum_micranthum GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum cassia GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum zeylanicum GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
************************************************************
Cinnamomum_camphora TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum_micranthum TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum cassia TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum_zeylanicum TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
************************************************************
Cinnamomum_camphora AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum_micranthum AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum cassia AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum zeylanicum AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
************************************************************
Cinnamomum_camphora AGACCTCGTGACTGGACTTCCAAAAATTTTCAAACAAAGAGGTATTTGATAAATTGAACG
Cinnamomum_micranthum AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGAGAAATTGAACG
Cinnamomum cassia AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGATAAATTGAACG
Cinnamomum_zeylanicum AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGCTAAATTGAACG
*********************************************************
Cinnamomum_camphora ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum_micranthum ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum cassia ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum zeylanicum ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
************************************************************
Cinnamomum_camphora TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum_micranthum TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum cassia TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum zeylanicum TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
************************************************************
Cinnamomum_camphora TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum_micranthum TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum cassia TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum zeylanicum TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
************************************************************
Cinnamomum_camphora GATTCATAGGCTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum_micranthum GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum cassia GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum_zeylanicum GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
***********************************************************
Cinnamomum_camphora CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum_micranthum CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum cassia CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum_zeylanicum CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
************************************************************
Cinnamomum_camphora TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum_micranthum TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum cassia TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum_zeylanicum TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
************************************************************
Cinnamomum_camphora CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum_micranthum CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum cassia CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum_zeylanicum CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
************************************************************
Cinnamomum_camphora AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum_micranthum AATCAAATGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum cassia AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum_verum AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
***********************************************************
Cinnamomum_camphora TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum_micranthum TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum cassia TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum zeylanicum TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
************************************************************
Cinnamomum_camphora AGTCCCCTTGGTTTACCTATATGAGTAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum_micranthum AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum cassia AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum zeylanicum AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
*********************************************************
Cinnamomum_camphora TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum_micranthum TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum cassia TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum zeylanicum TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
************************************************************
Cinnamomum_camphora ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum_micranthum ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum cassia ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum zeylanicum ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
************************************************************
Cinnamomum_camphora TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum_micranthum TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum cassia TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum zeylanicum TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
************************************************************
Cinnamomum_camphora CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum_micranthum CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum cassia CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum zeylanicum CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
************************************************************
Cinnamomum_camphora TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum_micranthum TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum cassia TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum zeylanicum TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
************************************************************
特异区段2
Cinnamomum_micranthum TTATAGGGATAGCAAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum zeylanicum TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum cassia TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum_camphora TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
***********************************************************
Cinnamomum_micranthum CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum zeylanicum CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum cassia CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum_camphora CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
************************************************************
Cinnamomum_micranthum TTTCACCATTCATTATTTTTTTATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum zeylanicum TTTCACCATTCATTATTTTTTAATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum cassia TTTCACCATTCATTATTTTTTGATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum_camphora TTTCACCATTCATTATTTTTTGATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
***********************************************************
Cinnamomum_micranthum TACAGATATTGTACATAAAACAAAATGTTGTACGTAAAATAAAAAAAAAAAAAGAAAAAT
Cinnamomum zeylanicum TACAGATATTGTACATAAAACAAAATGTTGTACGTAA---AAAAAAAAAAAAAGAAAAAT
Cinnamomum cassia TACAGATATTGTACATAAAACAAAATGTTGTACGTAAA--AAAAAAAAAAAAAGAAAAAT
Cinnamomum_camphora TACAGATATTGTACATAAGACAAAATGTTGTACGT-----AAAAAAAAAAAAAGAAAAAT
******************************************************
Cinnamomum_micranthum GCTTTGATTTTTTCCTAAAATCAAATTCTTTTGAAGAATAAGAGTATATAAATTGCAGGT
Cinnamomum zeylanicum GCTTTGATTTTTTCCTAAAATCAAATTATTTTGAAGAATAAGAGTATATAAAATGCAGGT
Cinnamomum cassia GCTTTGATTTTTTCAAAAAATCAAATTATTTTGAAGAATAAGAGTATATAAAATGCAGGT
Cinnamomum_camphora GCTTTGATTTTTTCAAAAAATCAAATTATTTTGAAGAATAAGAATATATAAAATGCAGGT
*******************************************************
Cinnamomum_micranthum TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum zeylanicum TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum cassia TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum_camphora TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
************************************************************
Cinnamomum_micranthum TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATAGATC
Cinnamomum zeylanicum TAAAAGAAATTAAAATGAAAAACGATTATGTGAATAAAACACTACTTAACCAAATGGATC
Cinnamomum cassia TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATAGATC
Cinnamomum_camphora TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATGGATC
*********************************************************
Cinnamomum_micranthum AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum zeylanicum AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum cassia AATACCAAACTTCTTGTTCTATTAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum_camphora AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
******************************************************
Cinnamomum_micranthum ACTAATACCAAAGTATTATCCATTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum zeylanicum ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum cassia ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum_camphora ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
***********************************************************
Cinnamomum_micranthum GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum zeylanicum GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum cassia GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum_camphora GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
************************************************************
Cinnamomum_micranthum ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum zeylanicum ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum cassia ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum_camphora ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
************************************************************
Cinnamomum_micranthum ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTGAACCAGATACCTACTAC
Cinnamomum zeylanicum ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTGAACCAGATACCTACTAC
Cinnamomum_cassia ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTAAACCAGATACCTACTAC
Cinnamomum_camphora ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTAAACCAGATACCTACTAC
***********************************************************
Cinnamomum_micranthum AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum zeylanicum AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGAGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum cassia AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum_camphora AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
***********************************************************
特异区段3
Cinnamomum_micranthum TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum zeylanicum TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum cassia TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum_camphora TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
************************************************************
Cinnamomum_micranthum AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum zeylanicum AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum cassia AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum_camphora AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
************************************************************
Cinnamomum_micranthum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum zeylanicum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum_aromaticum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum_camphora TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
************************************************************
Cinnamomum_micranthum ATAAGAGAGATATCTATCTGGATAGATCCATCAGAACGATTTGTAAATTAAGTGAGTAGG
Cinnamomum zeylanicum ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
Cinnamomum cassia ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
Cinnamomum_camphora ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
**********************************************************
Cinnamomum_micranthum GATTTATCCAATAACAAATATTTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum zeylanicum GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum cassia GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum_camphora GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
***********************************************************
Cinnamomum_micranthum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum zeylanicum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum_aromaticum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum_camphora GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
************************************************************
Cinnamomum_micranthum ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCATTGCACAGGGCTT
Cinnamomum zeylanicum ATATAGAGTAAAAAGACCCCCCCAAAAAGCCGTGCTTGCATTTTTCATTGCACAGGGCTT
Cinnamomum cassia ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCAGTGCACAGGGCTT
Cinnamomum_camphora ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCAGTGCACAGGGCTT
**********************************************************
Cinnamomum_micranthum TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum_zeylanicum TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum cassia TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum_camphora TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
************************************************************
Cinnamomum_micranthum CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATCAAATTTTTTTG
Cinnamomum zeylanicum CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
Cinnamomum cassia CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
Cinnamomum_camphora CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
*********************************************************
Cinnamomum_micranthum TGATTTTCTCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum zeylanicum TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum cassia TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum_camphora TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
***********************************************************
Cinnamomum_micranthum -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
Cinnamomum zeylanicum -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAACCCGTTCTCTAT
Cinnamomum cassia -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
Cinnamomum_camphora TTAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
**********************************************************
Cinnamomum_micranthum GTGACCTATGAGAAGGAGAAGAGGTTGTTGGAAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum zeylanicum GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum cassia GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum_camphora GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
***********************************************************
Cinnamomum_micranthum CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum zeylanicum CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum cassia CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum_camphora CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
************************************************************
Cinnamomum_micranthum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum zeylanicum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum_aromaticum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum_camphora TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
************************************************************
Cinnamomum_micranthum ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum zeylanicum ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum cassia ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum_camphora ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
************************************************************
Cinnamomum_micranthum CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum zeylanicum CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum cassia CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum_camphora CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
************************************************************
Cinnamomum_micranthum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum zeylanicum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum_aromaticum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum_camphora GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
************************************************************
Cinnamomum_micranthum CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum zeylanicum CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum cassia CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum_camphora CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
************************************************************
Cinnamomum_micranthum TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum zeylanicum TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum cassia TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum_camphora TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
************************************************************
Cinnamomum_micranthum GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGACATT
Cinnamomum zeylanicum GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGAGATT
Cinnamomum cassia GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGACATT
Cinnamomum_camphora GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTAAAAATGACATT
**********************************************************
Cinnamomum_micranthum GCCAAAAGTTGATAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum zeylanicum GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum cassia GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum_camphora GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
***********************************************************
Cinnamomum_micranthum CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum zeylanicum CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum cassia CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum_camphora CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
************************************************************
Cinnamomum_micranthum GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum zeylanicum GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum cassia GGGTTTTTTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum_camphora GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
***********************************************************
Cinnamomum_micranthum GTGTTCGCTCAAGAAAGGATCCAAAAGATTTGGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum zeylanicum GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum cassia GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum_camphora GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
***********************************************************
Cinnamomum_micranthum GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum zeylanicum GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum cassia GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum_camphora GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
***************************************************
特异区段4
Cinnamomum cassia TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum_camphora TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum zeylanicum TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum_micranthum TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
************************************************************
Cinnamomum cassia CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum_camphora CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum zeylanicum CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum_micranthum CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
************************************************************
Cinnamomum cassia TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum_camphora TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum zeylanicum TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum_micranthum TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
************************************************************
Cinnamomum cassia CTATTGTACCTTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum_camphora CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum zeylanicum CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum_micranthum CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
***********************************************************
Cinnamomum cassia TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_camphora TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_verum TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_micranthum TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
************************************************************
Cinnamomum cassia TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum_camphora TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum zeylanicum TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum_micranthum TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
************************************************************
Cinnamomum cassia CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG--AAAAAAAAAAAAAAAAATAATG
Cinnamomum_camphora CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG--AAAAAAAAAAAAAAAAATAATG
Cinnamomum zeylanicum CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAGAAAAAAAAAAAAAAAAAAATAATG
Cinnamomum_micranthum CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG-AAAAAAAAAAAAAAAAAATAATG
**********************************************************
Cinnamomum_cassia CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum_camphora CCTTCAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum zeylanicum CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum_micranthum CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
***********************************************************
Cinnamomum_cassia ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum_camphora ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum zeylanicum ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum_micranthum ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
************************************************************
Cinnamomum cassia TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum_camphora TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum zeylanicum TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum_micranthum TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
************************************************************
Cinnamomum cassia ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum_camphora ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum zeylanicum ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum_micranthum ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
************************************************************
Cinnamomum cassia CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum_camphora CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum zeylanicum CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum_micranthum CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
************************************************************
Cinnamomum cassia TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum_camphora TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum zeylanicum TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum_micranthum TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
************************************************************
Cinnamomum cassia TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_camphora TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_verum TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_micranthum TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
************************************************************
Cinnamomum cassia ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum_camphora ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum zeylanicum ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum_micranthum ACTTAATGAAAGTAGATTATCTTCCCATTC
****************************************************
步骤S8所述设计特异引物的方法为:使用引物设计软件(如:Primer3,Primer5,Primer blast等),在无差异的区域设计引物,且要求引物序列扩增差异区域。
通用PCR引物列表
步骤S9所述构建肉桂特异DNA分子标记矩阵的方法为:根据差异的SNP位点信息,选择肉桂与近缘种差异的SNP位点,按照基因组中的顺序,构建DNA分子标记矩阵(如表I~IV所示)。根据DNA矩阵的碱基位点,建立以下评分原则,待检测物种的碱基与矩阵碱基相同,矩阵中的物种得1分,不相同不得分也不减分。最终通过计算总得分,以及总得分与SNP位点的总分的比例,计算获得鉴定相似度。相似度越高,为矩阵中的物种的可能性越大,相似性大于95%,可判定为该物种。
实施例2
收集肉桂新鲜叶片,提取叶片的总DNA;提取的总DNA贮存于-20℃。设计引物序列如表1所示。以总DNA为模板进行PCR扩增;PCR反应体系见下表所示。
PCR反应体系(40μL)
PCR程序:预变性94℃5min;94℃30s,51℃30s,72℃1min20s,35个循环;72℃10min。
取3μL PCR扩增产物用1.2%的琼脂糖凝胶(EB染色)电泳检测。通过琼脂糖凝胶电泳回收大小对应的PCR产物片段,采用Sanger法测序。
将回收的片段进行Sanger测序;然后将测序结果SNP位点与差异结果进行比对鉴定,计算各序列相似性。
根据区段位点,统计各序列在DNA分子标记矩阵中的得分,碱基相同得一分,碱基不同不得分。矩阵总共包含65个DNA分子标记,总分65分。实施例中肉桂的鉴定率为100%。
实施例3
收集香樟的新鲜叶片,提取叶片的总DNA;提取的总DNA贮存于-20℃。设计引物序列如表2所示。以香樟总DNA为模板进行PCR扩增;PCR反应体系见下表所示。
PCR反应体系(40μL)
PCR程序:预变性94℃5min;94℃30s,51℃30s,72℃1min20s,35个循环;72℃10min。
取3μL PCR扩增产物用1.2%的琼脂糖凝胶(EB染色)电泳检测。通过琼脂糖凝胶电泳回收大小对应的PCR产物片段。
根据区段位点,统计各序列在DNA分子标记矩阵中的得分,碱基相同得一分,碱基不同不得分。矩阵总共包含65个DNA分子标记,总分65分。实施例中肉桂的鉴定率为52.31%,香樟的鉴定率为95.38%。鉴定物种是香樟,不是肉桂。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 广州中医药大学(广州中医药研究院)
<120> 肉桂及其近缘植物的鉴定或鉴别方法,和该方法所用的引物及试剂盒
<160> 8
<170> PatentIn version 3.3
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Claims (10)
1.引物组合产品,其包括下述引物对:
引物对P1:SEQ ID NO:1-2;
引物对P2:SEQ ID NO:3-4;
引物对P3:SEQ ID NO:5-6;以及
引物对P4:SEQ ID NO:7-8。
2.试剂盒,其含有权利要求1所述的引物组合产品。
3.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒还含有DNA提取体系、PCR反应缓冲液、水、DNA聚合酶以及分子量marker中的一种或多种。
4. 根据权利要求3所述的试剂盒,其特征在于,所述DNA聚合酶选自Taq、Bst、Vent、Phi29、Pfu、Tru、Tth、Tl1、Tac、Tne、Tma、Tih、Tf1、Pwo、Kod、Sac、Sso、Poc、Pab、Mth、Pho、ES4DNA聚合酶以及Klenow片段中的任一种。
5.根据权利要求3所述的试剂盒,其特征在于,所述水为无核酸酶的水。
6.根据权利要求2~5任一项所述的试剂盒,其特征在于,所述试剂盒还含有测序所需试剂。
7. 肉桂(Cinnamomumcassia)及其近缘植物的鉴定或鉴别方法,其特征在于,所述近缘植物为锡兰肉桂(Cinnamomum zeylanicum)、香樟(Cinnamomum camphora)和沉水樟(Cinnamomummicranthum)植物,所述方法包括:
a)获取待检测植物的基因组DNA或线粒体DNA;
b)使用权利要求1所述的引物组合产品或权利要求2~6任一项所述的试剂盒对a)所得DNA进行扩增;
c)将扩增所得片段测序并将测序结果与肉桂特异DNA分子标记矩阵进行比对,若与所述矩阵中某物种总相似性大于95%则判定所述待检测植物为该物种;
根据DNA矩阵的碱基位点,建立以下评分原则:待检测物种的碱基与矩阵碱基相同,矩阵中的物种得1分,不相同不得分也不减分,最终通过计算总得分以及总得分与SNP位点的总分的比例,计算获得鉴定相似度;
所述矩阵为:
表I 引物对P1所扩增区域包含的SNP
表II 引物对P2所扩增区域包含的SNP
续表II
表III 引物对P3所扩增区域包含的SNP
续表III
表IV 引物对P4所扩增区域包含的SNP
。
8.根据权利要求7所述的方法,其特征在于,在各引物对进行扩增时,退火温度均为49℃~53℃。
9.根据权利要求7所述的方法,其特征在于,所述测序选自高通量测序。
10.根据权利要求7所述的方法,其特征在于,所述测序选自Sanger测序。
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